The present invention relates to the field of molecular biology and particularly to methods for amplification of ribonucleic acid (RNA).
PAP Technology for Amplification of DNA Template
Pyrophosphorolysis activated polymerization is a method for nucleic acid amplification1 2 where pyrophosphorolysis and polymerization are serially coupled by DNA polymerase using 3′ blocked primers. A primer is blocked at the 3′ end with a non-extendable nucleotide (3′ blocker), such as a dideoxynucleotide, and cannot be directly extended by DNA polymerase. When the 3′ blocked primer anneals to its complementary DNA template, DNA polymerase can remove the 3′ blocker from the 3′ blocked primer in the presence of pyrophosphate, which reaction is called pyrophosphorolysis. The DNA polymerase can then extend the 3′ unblocked primer on the DNA template. In addition to references cited herein, PAP has been described in U.S. Pat. Nos. 6,534,269, 7,033,763, 7,105,298, 7,238,480, 7,504,221, 7,914,995, and 7,919,253.
The serial coupling of pyrophosphorolysis and extension using the 3′ blocked primer in PAP results in an extremely high selectivity1 3 because a significant nonspecific amplification (Type II error) requires mismatch pyrophosphorolysis followed by mis-incorporation by the DNA polymerase, an event with a frequency estimated to be 3.3×10−11.
The bi-directional form of PAP (Bi-PAP) is especially suitable for allele-specific amplification that uses two opposing 3′ blocked primers with a single-nucleotide overlap at their 3′ ends3 4. Bi-PAP can detect one copy of a mutant allele in the presence of 109 copies of the wildtype DNA without false positive amplifications.
PAP was initially tested with Tfl and Taq polymerases using DNA template of the human dopamine D1 gene1, proving the principle that DNA-dependent DNA pyrophosphorolysis and DNA-dependent DNA polymerization can be serially coupled. The efficiency of PAP was greatly improved using TaqFS, a genetically engineered polymerase containing contain a F667Y mutation5, which were demonstrated using other DNA templates2 4 6.
However, no evidence has showed that PAP can work using RNA template, a long-felt but unsolved need. For PAP to work using RNA template, it is also required RNA-dependent DNA polymerization and RNA-dependent DNA pyrophosphorolysis which latter feasibility has not been demonstrated.
RNA-Dependent DNA Polymerization or Reverse Transcription Used in RT-PCR
The first step of RT-PCR is DNA polymerization or primer extension on RNA template that is catalyzed by RNA-dependent DNA polymerase or reverse transcriptase. Avian myeloblastosis virus (AMV) and moloney murine leukemia virus (MMLV) reverse transcriptases, two mesophilic retroviral transcriptases, are commonly used in this first step to convert the RNA template into its complimentary DNA (cDNA) product7 8. Native Taq, a thermophilic DNA polymerase, has measurable reverse transcriptase activity particularly in the presence of Mn2+ divalent ion9. rTth, another thermophilic DNA polymerase, shows over 100-fold greater reverse transcriptase activity than Taq even though they have significant amino acid sequence similarity10. Furthermore, Taq and rTth polymerases were also genetically engineered in order for higher reverse transcriptase activity11 12 13 14.
RNA-Dependent DNA Pyrophosphorolysis
Taq, Tfl, TaqFS, Pfu, and Vent polymerases can catalyze DNA-dependent DNA pyrophosphorolysis1 2 3 15. HIV and HCV reverse transcriptases were also reported to catalyze DNA-dependent DNA pyrophosphorolysis that removes 3′ dideoxynucleotide from DNA primer on synthetic DNA (rather than RNA) template16 17.
However, there was no report of RNA-dependent DNA pyrophosphorolysis of polymerase that removes 3′ deoxyribonucleotide or 3′ dideoxynucleotide or 3′ acyclonucleotide from a primer using RNA as template.
Advantages of the Invention
It is advantageous that RNA-PAP can direct amplify RNA template without additional treatment. In addition, RNA-PAP has high selectivity against mismatches on the RNA template, providing highly specific amplification of RNA template. Furthermore, we genetically engineered mutant polymerases for higher RNA-PAP efficiency.